(1) Structural mechanism of multi-substrate selectivity in urea/amide channel (UAC) from Bacillus cereus UAC from B. cereus is highly permeable to both urea and nicotinamide, whose structures and sizes are different. This makes UAC an interesting target to study the mechanism of selectivity on multiple substrates. UAC is a hexameric membrane protein of 132 kDa with each protomer as an independent channel. It is fully embedded in the lipid membrane without a soluble domain. Its small size and complete embedding in the membrane also make it a challenging target for cryoEM structural study. Postdoc fellow Tengfei Lian in my group expressed and purified UAC from E. coli and performed structural and functional studies. We obtained the cryoEM structure of UAC in the apo form at 3.4 resolution. The structure shows that UAC hexamer has a disk-shaped structure with 6-fold rotational symmetry. Each protomer comprises of 7 transmembrane (TM) helices connected by short loops. In each protomer, 6 TM helices assemble the permeation pathway. We also obtained the cryoEM structure of UAC in complex with two nicotinamide molecules in the permeation pathway. Comparison between the apo and complex structures suggests some key residues important for substrate selectivity. Tengfei also did mutagenesis on these residues and activity assays confirm our hypothesis. We are still working on getting higher resolution structures using high-end cryo electron microscopes and more functional studies to elucidate the mechanism of substrate selectivity in UAC. (2) Structural study of anion exchanger 1 (AE1) AE1 (a.k.a. Band 3) is a member of the solute carrier (SLC) 4 family of bicarbonate transporters. It is the major membrane protein in red blood cells. It mediates exchange of bicarbonate and chloride ions across the cell membrane by its membrane domain and also participates in maintenance of the erythrocyte shape by binding to cytoskeletal proteins through its cytoplasmic domain. Mutations in AE1 are implicated in red cell diseases, including thalassemia, sickle cell anemia, and Southeast Asian ovalocytosis. We recently solved the cryoEM structure of human AE1 membrane domain (about 120 kDa), which is the transporter domain, at 3.4 resolution. Interpretation of this structure suggests that the transporter is in the inward-facing open state, different from the X-ray crystal structure of AE1 membrane domain in the outward-facing open state. Comparison between the structures in these two different open states suggests a plausible mechanism of ion transport in AE1. We also discovered some unexpected structural rearrangement in the inward-facing open state. (3) CryoEM structural study of small membrane proteins Postdoc fellow Xiaodan Ni in my group is pushing the limit of cryoEM study on small membrane proteins. She purified GadC, a 55 kDa glutamate-GABA antiporter, to test the feasibility of cryoEM study on such a small membrane protein. The size limit of cryoEM on membrane proteins is generally considered >150 kDa. The preliminary cryoEM data of GadC show good image contrast that is promising for high-resolution data processing. Xiaodan is currently working on optimizing sample preparation, cryoEM data collection and processing.